Pharmacological agents are being developed to modulate phosphotyrosyl (pTyr) dependent cell signalling. Emphasis is on inhibitors of pTyr dependent binding interactions which are mediated by src homology 2 (SH2) domains and on protein- tyrosine phosphatase (PTP) inhibitors. Central to both of these efforts is the development of new pTyr mimetics which afford either increased stability toward enzymatic degradation by PTPs or increased affinity. In the SH2 domain area, development of cell-permeable growth factor receptor-bound 2 (Grb2) antagonists is being undertaken as potential new therapeutics for a variety of cancers including erbB-2 and Met dependent cancers. Previously we had prepared a series of pTyr-mimicking amino acid analogues that were stable toward enzymatic degradation. These were incorporated into tripeptide platforms and shown to have high Grb2-SH2 domain binding affinity. Our efforts in this area have continued with the preparation of a new pTyr mimetic having both the labile phosphate ester bond replaced with a stable methylene group and the alpha-nitrogen functionality replaced with a carboxymethylene moiety. When incorporated into an appropriate tripeptide platform, this novel amino acid analogue exhibits superior potency in both extracellular and whole cell assays. During the reporting period novel macrocyclic peptide mimetics were prepared that represent conformationally constrained variants of our earlier tripeptide inhibitors. In extracellular Grb2-SH2 domain binding assays, these new macrocyclic compounds displayed significantly enhanced potency. For certain analogues, binding constants in the near picomolar range were observed. Importantly, in whole cell assays these macrocyclic ligands maintained enhanced potency. In vivo inhibition constants against Grb2 SH2 domain binding were in the sub-micromolar range. In culture against breast cancer cells mitogenically driven through erB-2 dependent pathways that utilize Grb2, these macrocycles displayed good cytostatic effects. The same compounds were non-toxic against similar cells that are not dependent on Grb2 pathways for survival. Collaborative studies are currently underway to examine these agents in combination therapies directed against breast cancer. Parallel collaborations are studying effects agents against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb2-dependent signaling pathways. The success of our macrocyclization strategy has lead us to explore alternate macrocyclization techniques. This has included novel aza-peptide macrocycles in which critical carbon atoms are replaced by nitrogens. In another approach, the first beta-amino pTyr mimetic yet reported was prepared and is currently being utilized to prepare a new family of beta-amido macrocyclized analogues.In the phosphatase area, a structure-based approach toward inhibitor design is being pursued. Using an epidermal growth factor receptor (EGFr)-derived pTyr-containing peptide sequence as a platform, we had previously delineated a number of novel non phosphorus containing pTyr mimetics for inhibitory potency against PTP1B. Highly potent motifs identified in this fashion have served as models for small molecule peptidomimetic design. During the reporting period a new effort was begun to develop inhibitors of YopH, a PTP active pathogenic component of Yersinia pestis. Such efforts are predicated on the need for protective agents against potential use of Yersinia pestis as a bioterrorism organism. To date using novel pTyr mimetics, tripeptide inhibitors have been identified that exhibit low micromolar affinity against YopH. Focussed libraries are in progress to optimize these leads. As part of this effort, a collaboration is in place determine X-ray crystal structures ofour inhibitors co-crystallized with the YopH enzyme. Information derived from these studies will be used for the structure-based design of peptidomimetic YopH inhibitors as potential protective agents against YopH exposure.